Hair-like pilus fibers found on many bacteria are believed responsible for virulent attachment to host epithelial cells. Recent human challenger studies on Neisseria gonorrhoeae have shown that only piliated cells are infectious, thus making pilin the only gonococcal outer membrane protein shown to be required for virulence. The overall goal of this competitive renewal is to finish the determination of the atomic structure of Neisseria gonorrhoeae pilin and its relationship to pilus self-assembly and function. The specific aims are to complete the determination of three-dimensional structure of the pilin protein subunit, the molecular basis for pilin self-association to form the pilus fiber, the location and identity of other proteins in the pilus, and the structural implications for the interaction of pilus proteins with the epithelial cell surface. Pilin crystals obtained during the first funding period diffract to atomic resolution making this the first fiber-forming protein whose atomic structure can be determined by x-ray diffraction. Results from immunoelectron microscopy and competitive immunoassays, using both polyclonal and monoclonal antibodies both to whole protein and to pilin peptide sequences, will be combined with x-ray diffraction data on single crystals of pilin and analyzed using both computational and computer graphic methods. Although no bacterial pilus structures are yet known, that knowledge is indispensable to understanding the basic protein chemistry and structure and function of the bacterial pilus and its role in the pathogenesis for gonorrhea and several other related bacterial diseases representing major threats to public health. The overall medical importance of this understanding is underlined by the ever increasing antibiotic resistance of pathogenic bacteria.